It has been know for decades that DNA or RNA can cross the plasma membrane into a cell and, once inside the cell, encoded protein(s) can be expressed. Likewise, the search for a nucleic acid transport mechanism has been ongoing for decades. Interest in the mechanism of uptake has been reinvigorated by the recent emergence of nucleic acid based therapeutics. This interest is due in part to the observation that the primary rate limiting steps in nucleic acid based therapy is poor nucleic acid uptake by cells. Understanding the nucleic acid transport mechanism is not only an important basic physiologic question, but in gaining this understanding, an important limitation of nucleic acid based therapy may be overcome. The long-term objective of the PI's laboratory, therefore, is to define and characterized the molecular mechanism of nucleic acid uptake by cells. We have chosen to use kidney transport of oligodeoxyribonucleicacid (ODN) as a model system. Kidney is an ideal model system because nucleic acids preferentially localize to kidney tissue following systemic administration, ODNs are transported into cells of the kidney where they are sequestered, and they are degraded very slowly within these cells. We have identified a protein complex, purified by ODN-affmity chromatography that functions as an ODN-conducting channel when reconstituted in model cell membranes in vitro. The complex is composed of a pore-forming subunit and a 36-kDa regulatory subunit (p36; see Preliminary Results). We have identified four protein components of the channel. One of which, cytosolic malate dehydrogenase, functions as the regulatory subunit of the channel. The role of the remaining three proteins has not been fully characterized. We have shown in LLC-PKL cells that the channel is an important part of ODN uptake and we have an abundance of data in lipid bilayer experiments demonstrating that cytosolic malate dehydrogenase is the regulatory subunit of this channel. A role for cMDH in ODN transport in living tissue, however, has not been shown. Therefore, the aim of the proposed studies is to test the hypotheses that 1) the nucleic acid channel is the primary route of nucleic acid transport in renal epithelium, and 2) cytosolic malate dehydrogenase participates in nucleic acid transport. We will test these hypothesis by addressing the following specific aims:1) To determine the relative contribution of the nucleic acid channel, endocytosis, and pinocytosis to total nucleic acid uptake in renal epithelium. 2) To determine the functional role of cytosolic malatedehydrogenase in nucleic acid transport; 3) To establish the topology of cMDH relative to plasma membrane.